It is well known that glutathione is a tripeptide thiol (L-.gamma.-glutamyl-L-cysteinylglycine; GSH) which is found in virtually all cells and functions in metabolism, transport, and cellular protection. GSH is a coenzyme for several enzymatic reactions; GSH is involved in the reduction of the disulfide linkages of proteins and other molecules, in the synthesis of deoxyribonucleotide precursors of DNA, in the protection of cells against the effects of free radicals and of reactive oxygen intermediates like peroxides, and in the transport of amino acids.
Modifications of glutathione metabolism can be achieved by the administration of selective enzyme inhibitors to decrease intracellular GSH levels, or by administration of compounds that increase GSH synthesis. Such effects are useful in chemotherapy and radiation therapy and in protecting cells against the toxic effects of drugs, other foreign compounds and oxygen, and in preventing the expression of latent HIV in AIDS. The many functions of GSH are important in many fields of biology enzymology and transport, pharmacology, radiation biology, cancer therapy, toxicology, endocrinology, microbiology, agriculture, virology, and immunology. The enzymology, metabolism and functions of GSH are outlined in Meister. "Metabolism and Function of Glutathione", in "Glutathione: Chemical, Biochemical, and Medical Aspects" Part A. pages 367-474, volume III of Coenzymes and Cofactors, eds. D. Dolphin, R. Poulseon, O. Avramovic; John Wiley, New York, 1989.
Modification of GSH metabolism to deplete or increase cellular GSH may serve various purposes. For example, it is well known that thiols protect cells against the effects of irradiation. Since decreasing cellular GSH levels makes cells more susceptible to irradiation, GSH depletion is useful in chemotherapeutic situations in which the cells to be destroyed and the cells to be spared have substantially different quantitative requirements for GSH. Depletion of GSH by inhibition of its synthesis also serves as a valuable adjuvant in chemotherapy with drugs that are detoxified by reactions involving GSH.
Development of resistance to a drug or to radiation therapy may be associated with an increase in cellular GSH. GSH is involved in the detoxification of many drugs, and it is known that a significant pathway of acetaminophen detoxification involved conjugation with GSH.
GSH is required for lymphocyte proliferation. Depletion of lymphocyte GSH by treatment with a selective inhibitor of GSH synthesis, L-buthionine-SR-sulfoximine (BSO), inhibits proliferation. Conversely, supplying GSH permits lymphocyte proliferation.
When lymphocytic cells containing HIV (a latent model of HIV infection ) are treated with thiols, such as N-acetyl cysteine, GSH and GSH monoethyl ester, and stimulated, they produce little virus in comparison with the cells that are not thiol treated.
Treatment with a thiazolidine such as L-2-oxothiazolidine-4-carboxylic acid, may be of value to patients with liver disease and to premature infants who may be deficient in the utilization of methionine sulfur for cysteine formation, and thus in GSH synthesis. The effectiveness of such a thiazolidine as an intracellular cysteine precursor depends on the presence of 5-oxoprolinase, an enzyme activity found in almost all animal cells. This enzyme also occurs in plants, suggesting that such a thiazolidine, and hence glutathione, may be useful as a safener in agriculture to protect crop plants against the toxic effects of herbicides.
Various methods are known to increase cellular levels of glutathione. GSH is composed of three amino acids: glutamic acid, cysteine, and glycine. Administration to animals of the amino acid precursors of GSH may produce an increase in cellular GSH; however, there is a limit to the effectiveness of this procedure. Concentration of cellular GSH are dependent on the supply of cysteine, which is derived from dietary protein and by trans-sulfuration from methionine in the liver. Administration of cysteine is not a good method for increasing GSH levels because cysteine is rapidly metabolized and it is also toxic. Administration to animals of compounds that are transported into cells and converted intracellularly into cysteine is sometimes useful in increasing cellular GSH. For example, the thiazolidine, L-2-oxothiazolidine-4-carboxylic acid, is transported into the cell, where it is converted by 5-oxoprolinase into L-cystine, which is rapidly used for GSH synthesis.
Another way in which tissue GSH concentration may be increased is by administration of .gamma.-glutamyl amino acid is transported intact and serves as a substrate of GSH synthetase. This method is effective for cells which have a .gamma.-glutamyl amino acid transport system. It is also known that administration of N-acetyl-L-cysteine increases tissue concentrations of GSH. This latter method requires a deacetylase and may be limited by toxicity of N-acetyl-L-cysteines.
That the administration of GSH itself might lead to increase GSH levels has also been considered. However, there is little published data that shows intact GSH enters cells. There are several reports on particular biological systems indicating that GSH itself is not transported into cells. A very little GSH may be transported into some cells, such as intestinal cells. However, the increase in cellular GSH sometimes found in some cells after the administration of GSH is due to (a) extracellular breakdown of GSH, (b) transport of free amino acids or dipeptides derived for GSH extracellularly, and (c) intracellular resynthesis of GSH.
These previous methods of increasing intracellular GSH levels are disadvantageous in the areas of efficiency, toxicity, limits on effective concentration obtainable, etc. as discussed heretofore. In addition, the known methods which depend on synthesis of GSH by increasing the supply of substrates to the synthetases involved, depend on the presence of the synthetases, one of which is subject to feedback inhibition by GSH.
The administration of GSH monoesters increases cellular GSH in many cells, without the need from the enzymes of GSH synthesis. It is effective in preventing acetaminophen and some heavy metal poisoning.
Accordingly, an object of the invention is to provide a method for increasing the intracellular levels of GSH by delivering intact GSH to cells, rather than its amino acid or dipeptide substrates.
A further object of the invention is to provide pure derivatives of GSH, the use thereof in GSH delivery and a method for obtaining such pure derivatives.
Another object of the invention is to provide a method of increasing intracellular levels of GSH which is highly efficient and which does not depend on the presence of synthetases.
A further object of the present invention is to provide a method for increasing the intracellular levels of GSH with toxic effects of other methods.
Still another object of the invention is to provide a method for efficiency and rapidly increasing cellular GSH levels for any purpose for which elevated GSH levels are desired in the prior art, such as for drug detoxification, cellular protection against oxygen and its metabolites such as peroxides, free radicals, or foreign compounds, etc, and in lymphocyte function, in the treatment of AIDS and other viral infections, etc.